PCR Detection of Microbial Pathogens PCR Detection of Microbial ...

74 Lübeck and Hoorfar
The latter include enterobacterial repetitive intergenic consensus PCR (ERIC-
PCR) (83), repetitive extragenic palindromic PCR (REP-PCR) (83), denaturing
gradient gel electrophoresis (DGGE), (84), terminal restriction fragment length
polymorphism (T-RFLP) (85), and amplified ribosomal DNA restriction analysis
(ARDRA) (86). Among whole genome fingerprinting PCR methods, random
amplified polymorphic DNA (RAPD) (87), and amplified fragment length
polymorphism (AFLP) (88) are most common, but also arbitrarily primed-PCR
(AP-PCR) (89) and universally primed PCR (UP-PCR) (90) are used.
RAPD, AP-PCR, and UP-PCR can all be used for typing of organisms without
previous knowledge of DNA sequences. The use of one single primer leads to
amplification of several DNA fragments randomly distributed throughout the
genome. AFLP makes use of restriction enzyme digestion and addition of adapters
for primer annealing before amplification of the DNA for differentiation. In
all these methods, multiple bands are produced, and the fragments are separated
by gel electrophoresis, providing direct analysis of polymorphism of different
isolates. UP-PCR differs from the more well-known RAPD and AP-PCR techniques
in the design of primers. The primers used in RAPD are short, usually
8–12-mers, with random sequence composition. The amplification is very sensitive
to reaction conditions, in particular the annealing temperature. Primers in
UP-PCR are usually 15–21-mers and have a unique design targeting mainly evolutionary
younger intergenic segments of the genome. Based on the UP-PCR technique,
different derivative methods have been developed. One of these, the species
identification method, is a cross-blot hybridization variant, in which hybridization
of UP-PCR products obtained from isolates of one species with the same
primer reveals DNA homology (91,92). The cross-blot hybridization variant has a
potential as a DNA array-based typing method (93). Another application is the
development of diagnostics of specific strains by identification of unique markers
that can be detected selectively by conversion of the marker into sequence characterized
amplified method (SCAR) and using pairwise combinations of selected
primers (SCAR primers) for amplification of a specific product (94).
Fingerprinting methods are not suitable for direct detection of target organisms
in complex diagnostic samples. These methods require pure cultures due
to the nature of the primers that allow amplification from almost any organisms.
The main advantage of the methods is their potential to differentiate very
similar isolates of the same species, which makes them suitable for studies of
microbial populations in molecular epidemiology.
6. Critical Parameters in PCR
6.1. Inhibition of DNA Amplification
Failure of DNA amplification due to the presence of inhibitory substances is
a common problem in PCR and may, in some cases, be the main cause of false